Bacterial and Liposomal Antigen Processing Exogenous particulate antigens (Ags), e.g. bacteria, are processed for presentation to CD8 T cells by class I MHC (MHC-I) molecules via "alternate" MHC-I Ag processing mechanisms. The goal of the proposed studies is to elucidate these processing mechanisms to provide understanding of the basis for CD8 T cell responses to bacteria and a foundation for new vaccination strategies to elicit protective CD8 T cell immunity. We will study the processing of Ags coupled to latex beads or expressed by bacteria (Mycobacterium tuberculosis, E. coli) by murine dendritic cells and macrophages, which may use different Ag processing mechanisms (cytosolic vs. vacuolar). Aim 1: Elucidate the mechanisms of alternate MHC-I Ag processing: the roles of TAP, tapasin, post-Golgi vacuolar compartments, stabilizing peptides and heat shock proteins. Aim 2. Localize the sites of peptide binding to MHC-I in alternate MHC-I Ag processing. For example, we will probe for specific peptide:MHC-I complexes directly in phagosomes by using subcellular fractionation and novel approaches including flow organellometry arid organelle Ag presentation assays. Aim 3: Study the modulation of alternate MHC-I Ag processing by "pathogen-associated molecular patterns" (PAMPs): e.g. CpG DNA, M. tuberculosis (MTB) 19 kD lipoprotein, double-stranded RNA (e.g. poly I:C) and LPS, including the roles of Toll-like receptors (TLRs), e.g. TLR 2, TLR 4, TLR 9, and signaling molecules: MyD88, IFN-gamma signaling components, JAK/STAT and suppressors of cytokine synthesis (SOCS) molecules. Aim 4. Determine the alternate MHC-I processing mechanisms for MTB, an intravacuolar pathogen of great clinical significance, and the ways that MTB may modulate this processing by blocking phagosome maturation or other functions. Significance: Greater understanding of the mechanisms whereby MTB and other pathogens are processed to generate CD8 T cell responses will provide improved understanding for development of vaccines or immunotherapies to combat tuberculosis and other infectious diseases.